Methods of treating NFA-1 organism infection using apocynin

ABSTRACT

Described herein are compounds and pharmaceutical formulations thereof that can be administered to a subject infected with or suspected of being infected with an organism having an Nfa-1 protein. Also provided herein are methods of treating an infection with an organism having an Nfa-1 protein in a subject in need thereof by administering a therapeutically effective amount of apocynin to the subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 61/940,683 filed on Feb. 17, 2014, having the title“Inhibitors Against NAD(P)H Oxidases and ROS Production Protect AgainstCytotoxicity of Pathogenic Amoebae,” the disclosure of which isincorporated herein in its entirety.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled 02152701.txt, created on Feb. 13, 2015 andhaving a size of 2,376 bytes. The content of the sequence listing isincorporated herein in its entirety.

BACKGROUND

Free-living amoebae can be pathogenic and cause severe infection thatcan result in debilitation or death. For some infections the death rateexceeds 95%. The high death tolls can be attributed, at least in part,to the lack of effective treatments. As such there exists a need for thedevelopment of improved treatments for amoebae infections.

SUMMARY

Pharmaceutical formulations containing an amount of a compound that caninhibit an Nfa-1 protein are provided. In some aspects, thepharmaceutical formulations contain a therapeutically effective amountof a compound according to formula I or a derivative thereof

to reduce cytoxicity in a subject suspected of being infected orinfected with an organism having an Nfa-1 protein, where R₁ can be N orC, R₂ is O can be H, R₃ is N can be C, R₄ can be H, OH, O, or S, R₅ canbe N or C, R₆ can be N or C, R₇ can be N or C, R₈ can be N or C, and R₉can be H or O. In some aspects, the compound of Formula I can be acompound according to Formula II, Formula III, Formula IV, or Formula V,or derivatives of compounds according to Formula II, Formula III,Formula IV, or Formula V. The organism having an Nfa-1 protein can be N.fowleri, N. gruberi, Acanthamoeba hatchetti, A. healyi, A. polyphaga, A.rhysodes, A. astronyxis, A. divionensis, A castellanii, Entamoebahistolytica, E. invadens, E. dispar, Balamuthia mandrillaris, Sappiniadiploidea, Pyrococcus furiosus, Clostridium acetobutylicum,Desulfovibrio vulgaris, Burkholderia pseudomallei, Desulfurococcusmucosus, Methanococcus jannaschii, Riftia pachyptila, Phascolopsisgouldii, Periserrula leucophryna, Perinereis aibuhitensis, Theromyzontessulatum, Hirudo medicinalis, or Themiste zostericola. Thetherapeutically effective amount of Formula I, Formula II, Formula III,Formula IV, Formula V or a derivative of any of the foregoing compoundscan, in some aspects, can reduce the oxidoreductase activity of theNfa-1 protein as compared to a control in the subject suspected of beinginfected or infected with the organism having the Nfa 1 protein. Inadditional aspects, the therapeutically effective amount of Formula I,Formula II, Formula III, Formula IV, Formula V or a derivative of any ofthe foregoing compounds can reduce the amount of reactive oxygen speciesin the subject suspected of being infected or infected with the organismhaving an Nfa-1 protein as compared to a control.

In further aspects, the pharmaceutical formulations contain atherapeutically effective amount of a compound to formula VI or aderivative thereof

to reduce cytoxicity in a subject suspected of being infected orinfected with an organism having an Nfa-1 protein, where R₁ can be H orOH, and R₂ can be H, COCH₃, or COH. In other aspects, the compoundaccording to formula VI can be a compound according to Formula VII,Formula VIII, Formula IX, or Formula X, or derivatives of compoundsaccording to Formula VII, Formula VIII, Formula IX, or Formula X. Theorganism having an Nfa-1 protein can be N. fowleri, N. gruberi,Acanthamoeba hatchetti, A. healyi, A. polyphaga, A. rhysodes, A.astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola. The therapeutically effectiveamount of Formulas VI, Formula VII, Formula VIII, Formula IX, or FormulaX, or derivatives of any of the foregoing compounds can, in someaspects, can reduce the oxidoreductase activity of the Nfa-1 protein ascompared to a control in the subject suspected of being infected orinfected with the organism having the Nfa 1 protein. In additionalaspects, the therapeutically effective amount of Formulas VI, FormulaVII, Formula VIII, Formula IX, or Formula X, or derivatives of any ofthe foregoing compounds can reduce the amount of reactive oxygen speciesin the subject suspected of being infected or infected with the organismhaving an Nfa-1 protein as compared to a control.

Methods of using the pharmaceutical formulations containing an amount ofa compound that can inhibit an Nfa-1 protein are provided. In someaspects, the method contains the step of administering a therapeuticallyeffective amount of Formula I or a derivative thereof:

to a subject in need thereof, where the subject in need thereof isinfected with or is suspected of being infected with at least oneorganism having an Nfa-1 protein, and where R₁ is N or C, R₂ is O or H,R₃ is N or C, R₄ is H, OH, O, or S, R₅ is N or C, R₆ is N or C, R₇ is Nor C, R₈ is N or C, and R₉ is H or O. In further aspects, thetherapeutically effective amount adminstered can reduce cytotoxicity inthe subject in need thereof as compared to a control. Thetherapeutically effective amount, in other aspects, can the amount ofreactive oxygen species in the subject in need thereof as compared to acontrol. In additional aspects, the therapeutically effective amount canreduce the oxidoreductase activity of the Nfa-1 protein as compared to acontrol. The organism having the Nfa-1 protein can be N. fowleri, N.gruberi, Acanthamoeba hatchetti, A. healyi, A. polyphaga, A. rhysodes,A. astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola. In some aspects of the method, thecompound of Formula I can be a compound according to Formula II, FormulaIII, Formula IV, Formula V or a derivative of any of the foregoingcompounds.

In other aspects, the method contains the step of administering atherapeutically effective amount of Formula IV or a derivative thereof:

to a subject in need thereof, where the subject in need thereof isinfected with or is suspected of being infected with at least oneorganism having an Nfa-1 protein, and R₁ can be H or OH, and R₂ can beH, COCH₃, or COH. In further aspects, the therapeutically effectiveamount adminstered can reduce cytotoxicity in the subject in needthereof as compared to a control. The therapeutically effective amount,in other aspects, can reduce the amount of reactive oxygen species inthe subject in need thereof as compared to a control. In additionalaspects, the therapeutically effective amount can reduce theoxidoreductase activity of the Nfa-1 protein as compared to a control.The organism having the Nfa-1 protein can be N. fowleri, N. gruberi,Acanthamoeba hatchetti, A. healyi, A. polyphaga, A. rhysodes, A.astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola. In some aspects of the method, thecompound of Formula VI can be a compound according to Formula VII,Formula VIII, Formula IX, Formula X or a derivative of any of theforegoing compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows a phylogram of N. fowleri Nfa-1 Hemerythrin (Hr) homologuesin amoebae, bacteria, archaea and polychaetes for which genes werepresent in the NCBI GeneBank database as of the filing date of U.S.Provisional application. Amino acid sequences of hemerythrin-relatedproteins from Naegleria amoeba, bacteria, archaea, and polychaete wormswere aligned using the ClustalW2 program.

FIG. 2 shows a phylogram of N. fowleri Nfa-1 Hemerythrin (Hr) andrelated proteins in other amoebae with sequenced genomes. Amino acidsequences of hemerythrin-related proteins from Naegleria amoebae (N.fowleri, N. gruberi) and the pathogenic amoebae Acanthamoeba (A.castellanii) and Entamoeba (E. invadens, E. dispar) were aligned usingthe ClustalW2 program. Protein IDs and accession numbers are given insquare brackets.

FIG. 3 demonstrates the results of a LIGPLOT analysis of N. fowleriNfa-1 Fe-oxygen Protein ligand interactions. LIGPLOT analysis of N.fowleri Nfa-1 bound with two ferric (Fe) ions and oxygen was generatedfrom the LIGPLOT and crystal structure of P. gouldii Hr (PDB: li4y at1.8 Å). The Hr-Fe binding site was made of five histidine (His) residuesand two acidic residues (glutamate, Glu60 and aspartate, Asp112) thatform ionic bonds (in blue) with two Fe ions. Phenylalanine Phe57 formshydrophobic interactions (depicted as eyelashes) that can stabilize theFe binding pocket. Hydrogen bonds and their lengths in Angstrom (Å) areshown in red.

FIG. 4 demonstrates a LIGPLOT analysis of an Acanthamoeba protein withan Nfa-1 like Hemerythrin domain as determined by a BLAST search of N.fowleri Nfa-1 against Acanthamoeba castellanii (Gen Bank Accession No.XP_004356500.1).

FIG. 5 demonstrates an alignment of N. fowleri Nfa-1 and an Acanthamoebacastellanii protein (Gen Bank Accession No. XP_004356500.1) with anNfa-1 hemerythrin-like domain. The asterik (*) indicates amino acidsthat make up the Fe-oxygen binding site of three-dimensional (3-D)protein, of which a model is shown in FIG. 6A-6B.

FIGS. 6A-6B demonstrate a side view (FIG. 6A) and a top view (FIG. 6B)of a 3-D ribbon diagram of N. fowleri Nfa-1 Hr. The labeled amino acidscorrespond to the (*) labeled amino acids of FIG. 5.

FIGS. 7A-7B show graphs demonstrating the results of an enzyme activityassay performed to determine enzyme kinetics of N. fowleri Nfa-1 Hr whenNADH (FIG. 7A) or NADPH (FIG. 7B) is used as a substrate.

FIG. 8 shows a table that summarizes the catalytic rates (k_(cat)),substrate affinities (K_(m)), catalytic efficiencies (k_(cat)/K_(m)),and maximum enzyme activities (V_(max)) of the Naegleria fowleri Nfa-1hemerythrin enzyme as determined from the enzyme kinetic assay of FIG.7A-7B.

FIG. 9 is a graph demonstrating an inhibitor profile and pharmacology ofN. fowleri Nfa-1 Hr (oxidase) in vitro. Enzyme activity in the presenceof various substrate analogues and inhibitors were determined, includingthe NADH substrate analogue hypoxanthine (K_(i)=1 mM), the xanthineoxidase inhibitor Allopurinol (K_(i)=134 μM), the NAD(P)H oxidaseinhibitors DPI (diphenyleneiodonium chloride) (K_(i)=19 μM) and Apocynin(K_(i)=17 μM), the NADH peroxidase inhibitors AgNO₃ (K_(i)=345 μM) andpHMB (para-hydroxymercuribenzoate) (K_(i)=78 μM), and the NADHperoxidase activator H₂O₂ (9.7% stimulation).

FIG. 10 is a graph demonstrating the effect of various compounds on thecytotoxicity of N. fowleri in vitro. Cytotoxicity of N. fowleri amoebaewas measured with CHO cells co-cultured with N. fowleri for 13 hours at37° C. using the LDH cytotoxicity assay (infection ratio of 1 amoebacell per 5 CHO cells). Strong protection from the high levels of N.fowleri cytotoxicity in vivo was found for the oxidase inhibitorsApocynin (IC₅₀=15 μM), Allopurinol (IC₅₀=48 μM), DPI (IC₅₀=49 μM), andhypoxanthine (IC₅₀=497 μM).

FIGS. 11A-11B show graphs demonstrating the production of reactiveoxygen species (ROS) (FIG. 11A) or hydrogen peroxide (FIG. 11B).

FIGS. 12A-12B show the amino acid sequence (SEQ ID NO: 3) (FIG. 12A) andthe genomic DNA sequence (SEQ ID NO: 4) (FIG. 12B) for N. fowleri Nfa-1.The underlined amino acids (FIG. 12A) and nucleotides (FIG. 12B)correspond to the Nfa-1 Fe-oxygen binding site as demonstrated in FIGS.3, 5, and 6A-6B. The first Met in SEQ ID NO: 3 corresponds to the firstthree nucleotides in SEQ ID NO: 4 (the “start” codon). The last threenucleotides (“TAA”) of SEQ ID NO: 4 correspond to the “stop” codon andthus do not have a corresponding amino acid in SEQ ID NO: 3.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, biochemistry, botany and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

DEFINITIONS

The term “biocompatible”, as used herein, refers to a material thatalong with any metabolites or degradation products thereof that aregenerally non-toxic to the recipient and do not cause any significantadverse effects to the recipient. Generally speaking, biocompatiblematerials are materials which do not elicit a significant inflammatoryor immune response when administered to a patient.

The term “molecular weight”, as used herein, generally refers to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (M_(w)) as opposed to the number-average molecularweight (M_(n)). Capillary viscometry provides estimates of molecularweight as the inherent viscosity determined from a dilute polymersolution using a particular set of concentration, temperature, andsolvent conditions.

The term “biodegradable” as used herein, generally refers to a materialthat will degrade or erode under physiologic conditions to smaller unitsor chemical species that are capable of being metabolized, eliminated,or excreted by the subject. The degradation time is a function ofcomposition and morphology. Degradation times can be from hours toweeks.

The term “hydrophilic”, as used herein, refers to substances that havestrongly polar groups that readily interact with water.

The term “hydrophobic”, as used herein, refers to substances that lackan affinity for water; tending to repel and not absorb water as well asnot dissolve in or mix with water.

The term “lipophilic”, as used herein, refers to compounds having anaffinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combininghydrophilic and lipophilic (hydrophobic) properties.

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within +1-10% of the indicated value, whichever is greater.

As used herein, “control” or “suitable control” is an alternativesubject or sample used in an experiment for comparison purposes andincluded to minimize or distinguish the effect of variables other thanan independent variable. A “control” can be positive or negative. A“control” as used herein refers to a control that will allowdetermination of a response to a compound, formulation, or treatmentregimen described herein. “Control” includes a level of a physiologiccharacteristic or other parameter in a subject to be treated beforeadministration of a compound or formulation described herein or before atreatment regimen. “Control” includes a pre-made standard or a range ofvalues pre-determined to represent a normal level of the parameter beingmeasured in a subject.

As used herein, “concentrated” used in reference to an amount of amolecule, compound, or composition, including, but not limited to, achemical compound, polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, that indicates that the sample isdistinguishable from its naturally occurring counterpart in that theconcentration or number of molecules per volume is greater than that ofits naturally occurring counterpart.

As used herein, “diluted” used in reference to a an amount of amolecule, compound, or composition including but not limited to, achemical compound, polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, that indicates that the sample isdistinguishable from its naturally occurring counterpart in that theconcentration or number of molecules per volume is less than that of itsnaturally occurring counterpart.

As used herein, “purified” is used in reference to a nucleic acidsequence, peptide, or polypeptide or other compound that has increasedpurity relative to the natural environment or the environment in whichit was produced in.

The terms “sufficient” and “effective”, as used interchangeably herein,refer to an amount (e.g. mass, volume, dosage, concentration, and/ortime period) needed to achieve one or more desired result(s). Forexample, a therapeutically effective amount refers to an amount neededto achieve one or more therapeutic effects.

As used herein, “pharmaceutical formulation” refers to the combinationof an active agent, compound, or ingredient with a pharmaceuticallyacceptable carrier or excipient, making the composition suitable fordiagnostic, therapeutic, or preventive use in vitro, in vivo, or exvivo.

As used herein, “pharmaceutically acceptable carrier or excipient”refers to a carrier or excipient that is useful in preparing apharmaceutical formulation that is generally safe, non-toxic, and isneither biologically or otherwise undesirable, and includes a carrier orexcipient that is acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier or excipient”as used in the specification and claims includes both one and more thanone such carrier or excipient.

As used herein, “pharmaceutically acceptable salt” refers to any acid orbase addition salt whose counter-ions are non-toxic to the subject towhich they are administered in pharmaceutical doses of the salts.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” includes a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term farmanimal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama,alpaca, turkey, and the like.

As used herein, “therapeutic” refers to treating or curing a disease orcondition.

As used herein, “preventative” refers to hindering or stopping a diseaseor condition before it occurs in a subject or while the disease orcondition is still in the sub-clinical phase.

As used herein, “separated” refers to the state of being physicallydivided from the original source or population such that the separatedcompound, agent, particle, chemical compound, or molecule can no longerbe considered part of the original source or population.

As used herein, “active agent” or “active ingredient” refers to acomponent or components of a composition to which the whole or part ofthe effect of the composition is attributed.

As used herein, “tangible medium of expression” refers to a medium thatis physically tangible and is not a mere abstract thought or anunrecorded spoken word. Tangible medium of expression includes, but isnot limited to, words on a cellulosic or plastic material or data storedon a suitable device such as a flash memory or CD-ROM.

As used herein, “chemotherapeutic agent” or “chemotherapeutic” refer toa therapeutic agent utilized to prevent or treat cancer.

As used herein, “dose,” “unit dose,” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of the Nfa-1 inhibitor, derivative thereof, orformulation thereof calculated to produce the desired response orresponses in association with its administration.

As used herein, “antibody” refers to a protein produced by B cells thatis used by the immune system to identify and neutralize foreigncompounds, which are also known as antigens. Antibodies areglycoproteins belonging to the immunoglobulin superfamily. Antibodies,recognize and bind to specific epitopes on an antigen.

As used herein, “aptamer” refers to single-stranded DNA or RNA moleculesthat can bind to pre-selected targets including proteins with highaffinity and specificity. Their specificity and characteristics are notdirectly determined by their primary sequence, but instead by theirtertiary structure.

As used herein, “specific binding partner” or “binding partner” is acompound or molecule to which a second compound or molecule binds with ahigher affinity than all other molecules or compounds.

As used herein, “specifically binds” or “specific binding” refers tobinding that occurs between such paired species such asenzyme/substrate, receptor/agonist or antagonist, antibody/antigen,lectin/carbohydrate, oligo DNA primers/DNA, enzyme or protein/DNA,and/or RNA molecule to other nucleic acid (DNA or RNA) or amino acid,which may be mediated by covalent or non-covalent interactions or acombination of covalent and non-covalent interactions. When theinteraction of the two species produces a non-covalently bound complex,the binding that occurs is typically electrostatic, hydrogen-bonding, orthe result of lipophilic interactions. Accordingly, “specific binding”occurs between a paired species where there is interaction between thetwo which produces a bound complex having the characteristics of anantibody/antigen, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/protein,RNA/protein, RNA/amino acid, receptor/substrate interaction. Inparticular, the specific binding is characterized by the binding of onemember of a pair to a particular species and to no other species withinthe family of compounds to which the corresponding member of the bindingmember belongs. Thus, for example, an antibody preferably binds to asingle epitope and to no other epitope within the family of proteins.

As used herein “immunomodulator,” refers to an agent, such as atherapeutic agent, which is capable of modulating or regulating one ormore immune function or response.

As used herein, “differentially expressed,” refers to the differentialproduction of RNA, including, but not limited to, mRNA, tRNA, miRNA,siRNA, snRNA, and piRNA transcribed from a gene or regulatory region ofa genome or the protein product encoded by a gene as compared to thelevel of production of RNA by the same gene or regulator region in anormal or a control cell. In another context, “differentiallyexpressed,” also refers to nucleotide sequences or proteins in a cell ortissue which have different temporal and/or spatial expression profilesas compared to a normal or control cell.

As used herein, “overexpressed” or “overexpression” refers to anincreased expression level of an RNA or protein product encoded by agene as compared to the level of expression of the RNA or proteinproduct in a normal or control cell.

As used herein, “underexpressed” or “underexpression” refers todecreased expression level of an RNA or protein product encoded by agene as compared to the level of expression of the RNA or proteinproduct in a normal or control cell.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into RNA transcripts. In the context ofmRNA and other translated RNA species, “expression” also refers to theprocess or processes by which the transcribed RNA is subsequentlytranslated into peptides, polypeptides, or proteins.

As used herein, “isolated” means separated from constituents, cellularand otherwise, in which the polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, are normally associated with innature. A non-naturally occurring polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, do not require “isolation” todistinguish it from its naturally occurring counterpart.

As used herein, “nucleic acid” and “polynucleotide” generally refer to astring of at least two base-sugar-phosphate combinations and refers to,among others, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.“Polynucleotide” and “nucleic acids” also encompasses such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. For instance, theterm polynucleotide includes DNAs or RNAs as described above thatcontain one or more modified bases. Thus, DNAs or RNAs comprisingunusual bases, such as inosine, or modified bases, such as tritylatedbases, to name just two examples, are polynucleotides as the term isused herein. “Polynucleotide” and “nucleic acids” also includes PNAs(peptide nucleic acids), phosphorothioates, and other variants of thephosphate backbone of native nucleic acids. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, but contain the same bases. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “nucleic acids” or“polynucleotide” as that term is intended herein.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid(RNA)” generally refer to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA(small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA),anti-sense RNA, RNAi (RNA interference construct), siRNA (shortinterfering RNA), or ribozymes.

As used herein, “nucleic acid sequence” and “oligonucleotide” alsoencompasses a nucleic acid and polynucleotide as defined above.

As used herein, “DNA molecule” includes nucleic acids/polynucleotidesthat are made of DNA.

As used herein, “variant” refers to a polypeptide that differs from areference polypeptide or compound, but retains essential properties. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moremodifications (e.g., substitutions, additions, and/or deletions). Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. A variant of a polypeptide may be naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. A typical variant of a compound can be aderivative or analogue thereof.

As used herein, “functional variant” refers to a variant of a protein,polypeptide, molecule or compound (e.g., a variant of folic acid or afolic acid receptor protein) that can perform the same functions oractivities as the original protein or polypeptide, although notnecessarily at the same level (e.g., the variant may have enhanced,reduced or changed functionality, so long as it retains the basicfunction).

As used herein, “anti-infective” refers to compounds or molecules thatcan either kill an infectious agent or inhibit it from spreading.Anti-infectives include, but are not limited to, antibiotics,antibacterials, antifungals, antivirals, and antiprotozoans.

As used herein, “wild-type” is the typical form of an organism, variety,strain, gene, protein, or characteristic as it occurs in nature, asdistinguished from mutant forms that may result from selective breedingor transformation with a transgene.

As used herein “induces,” “inducing,” or “induced” refers to activatingor stimulating a process or pathway within a cell, such as endocytosis.

As used herein “heterogeneous” refers to a population of molecules,including nanoparticles, proteins, and polypeptides, or a population ofsubunits of a molecule that contains at least 2 molecules or subunitsthat are different from one another.

As used herein “homogenous” refers to a population of molecules,including nanoparticles, proteins, and polypeptides, or a population ofsubunits of a molecule in which all the molecules or subunits areidentical to one another.

As used herein, “derivative” refers to any compound having the same or asimilar core structure to the compound but having at least onestructural difference, including substituting, deleting, and/or addingone or more atoms or functional groups. The term “derivative” does notmean that the derivative is synthesized from the parent compound eitheras a starting material or intermediate, although this may be the case.The term “derivative” can include prodrugs, or metabolites of the parentcompound. Derivatives include compounds in which free amino groups inthe parent compound have been derivatized to form amine hydrochlorides,p-toluene sulfoamides, benzoxycarboamides, t-butyloxycarboamides,thiourethane-type derivatives, trifluoroacetylamides,chloroacetylamides, or formamides. Derivatives include compounds inwhich carboxyl groups in the parent compound have been derivatized toform methyl and ethyl esters, or other types of esters or hydrazides.Derivatives include compounds in which hydroxyl groups in the parentcompound have been derivatized to form O-acyl or O-alkyl derivatives.Derivatives include compounds in which a hydrogen bond donating group inthe parent compound is replaced with another hydrogen bond donatinggroup such as OH, NH, or SH. Derivatives include replacing a hydrogenbond acceptor group in the parent compound with another hydrogen bondacceptor group such as esters, ethers, ketones, carbonates, tertiaryamines, imine, thiones, sulfones, tertiary amides, and sulfides.“Derivatives” also includes extensions of the replacement of thecyclopentane ring with saturated or unsaturated cyclohexane or othermore complex, e.g., nitrogen-containing rings, and extensions of theserings with side various groups.

As used herein, “administering” refers to an administration that isoral, topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intracranial, intra-joint, parenteral, intra-arteriole,intradermal, intraventricular, intracranial, intraperitoneal,intralesional, intranasal, rectal, vaginal, intraurethral, byinhalation, or via an implanted reservoir. The term “parenteral”includes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,and intracranial injections or infusion techniques.

As used herein, “composition” refers to a combination of an activeagent(s) and another compound or composition, inert (for example, adetectable agent or label) or active, such as an adjuvant.

As used herein, “suitable substituent” means a chemically andpharmaceutically acceptable group, i.e., a moiety that does notsignificantly interfere with the preparation of or negate the efficacyof the inventive compounds. Such suitable substituents may be routinelychosen by those skilled in the art. Suitable substituents include butare not limited to the following: a halo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkynyl, C₃-C₈cycloalkenyl, (C₃-C₈ cycloalkyl)C₁-C₆ alkyl, (C₃-C₈ cycloalkyl)C₂-C₆alkenyl, (C₃-C₈ cycloalkyl) C₁-C₆ alkoxy, C₃-C₇ heterocycloalkyl, (C₃-C₇heterocycloalkyl)C₁-C₆ alkyl, (C₃-C₇ heterocycloalkyl)C₂-C₆ alkenyl,(C₃-C₇ heterocycloalkyl)C₁-C₆ alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C₁-C₆ alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C₁-C₆alkylamino, di-(C₁-C₆ alkyl)amino, carbamoyl, (C₁-C₆ alkyl)carbonyl,(C₁-C₆ alkoxy)carbonyl, (C₁-C₆ alkyl)aminocarbonyl, di-(C₁-C₆alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C₁-C₆alkyl)sulfonyl, and arylsulfonyl. The groups listed above as suitablesubstituents are as defined hereinafter except that a suitablesubstituent may not be further optionally substituted.

As used herein, “optically substituted” indicates that a group may beunsubstituted or substituted with one or more substituents as definedherein.

The term “alkyl” refers to the radical of saturated aliphatic groups(i.e., an alkane with one hydrogen atom removed), includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,and C₃-C₃₀ for branched chains). In other embodiments, a straight chainor branched chain alkyl contains 20 or fewer, 15 or fewer, or 10 orfewer carbon atoms in its backbone. Likewise, in some embodimentscycloalkyls have 3-10 carbon atoms in their ring structure. In some ofthese embodiments, the cycloalkyl have 5, 6, or 7 carbons in the ringstructure.

The term “alkyl” (or “lower alkyl”) as used herein is intended toinclude both “unsubstituted alkyls” and “substituted alkyls,” the latterof which refers to alkyl moieties having one or more substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons in its backbone structure. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S— alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can besubstituted as defined above for alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy,” as used herein, refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl is an ether or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O—alkynyl. The terms“aroxy” and “aryloxy”, as used interchangeably herein, can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

The terms “amine” and “amino” (and its protonated form) areart-recognized and refer to both unsubstituted and substituted amines,e.g., a moiety that can be represented by the general formula:

wherein R, R′, and R″ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R_(C) or R and R′ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R_(C) represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R or R′ can bea carbonyl, e.g., R, R′ and the nitrogen together do not form an imide.In other embodiments, the term “amine” does not encompass amides, e.g.,wherein one of R and R′ represents a carbonyl. In further embodiments, Rand R′ (and optionally R″) each independently represent a hydrogen, analkyl or cycloakly, an alkenyl or cycloalkenyl, or alkynyl. Thus, theterm “alkylamine” as used herein means an amine group, as defined above,having a substituted (as described above for alkyl) or unsubstitutedalkyl attached thereto, i.e., at least one of R and R′ is an alkylgroup.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R and R′ are as defined above.

As used herein, “Aryl” refers to C₅-C₁₀-membered aromatic, heterocyclic,fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ringsystems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-,8-, 9-, and 10-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with one or more substituents including,but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, andcombinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl.”

The term “aralkyl,” as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aralkyloxy” can be represented by —O-aralkyl, wherein aralkylis as defined above.

The term “carbocycle,” as used herein, refers to an aromatic ornon-aromatic ring(s) in which each atom of the ring(s) is carbon.

“Heterocycle” or “heterocyclic,” as used herein, refers to a monocyclicor bicyclic structure containing 3-10 ring atoms, and in someembodiments, containing from 5-6 ring atoms, wherein the ring atoms arecarbon and one to four heteroatoms each selected from the followinggroup of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or isH, O, (C₁-C₁₀) alkyl, phenyl or benzyl, and optionally containing 1-3double bonds and optionally substituted with one or more substituents.Examples of heterocyclic rings include, but are not limited to,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.Heterocyclic groups can optionally be substituted with one or moresubstituents at one or more positions as defined above for alkyl andaryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R and R′are as defined above. Where X is an oxygen and R or R′ is not hydrogen,the formula represents an “ester”. Where X is an oxygen and R is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R is a hydrogen, the formula represents a “carboxylicacid.” Where X is an oxygen and R′ is hydrogen, the formula represents a“formate.” In general, where the oxygen atom of the above formula isreplaced by sulfur, the formula represents a “thiocarbonyl” group. WhereX is a sulfur and R or R′ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R is not hydrogen, the above formula represents a“ketone” group. Where X is a bond, and R is hydrogen, the above formularepresents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Exemplary heteroatoms include, but are notlimited to, boron, nitrogen, oxygen, phosphorus, sulfur, silicon,arsenic, and selenium.

As used herein, the term “nitro” refers to —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” refers to —SH; theterm “hydroxyl” refers to —OH; and the term “sulfonyl” refers to —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, e.g. 1-14 carbon atoms,and optionally include one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats.Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

Heteroatoms, such as nitrogen, may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e., a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

As used herein, “therapeutically effective amount” refers to the amountof an Nfa-1 inhibitor, derivative thereof, pharmaceutical formulationthereof, auxiliary agent, or secondary agent described herein that willelicit the biological or medical response of a tissue, system, animal,or human that is being sought by the researcher, veterinarian, medicaldoctor or other clinician. “Therapeutically effective amount” includesthat amount of Nfa-1 inhibitor, derivative thereof, or pharmaceuticalformulation thereof that, when administered alone or co-administeredwith a secondary agent, is sufficient to prevent development of, reduceor alleviate to some extent, one or more of the symptoms of an infectionof an organism having an Nfa-1 protein. “Therapeutically effect amount”includes that amount of Nfa-1 inhibitor, derivative thereof, orpharmaceutical formulation thereof that, when administered alone orco-administered with a secondary agent, is sufficient to reducecytotoxicity in a subject suspected of being infected with or infectedwith an organism having an Nfa-1 protein as compared to a control.“Therapeutically effect amount” includes that amount of Nfa-1 inhibitor,derivative thereof, or pharmaceutical formulation thereof that, whenadministered alone or co-administered with a secondary agent, issufficient to reduce reactive oxygen species (ROS) in a subject or cellthereof suspected of being infected with or infected with an organismhaving an Nfa-1 protein as compared to a control. “Therapeuticallyeffect amount” includes that amount of Nfa-1 inhibitor, derivativethereof, or pharmaceutical formulation thereof that, when administeredalone or co-administered with a secondary agent, is sufficient to reduceNfa-1 oxidoreductase activity of Nfa-1 in a subject suspected of beinginfected with or infected with an organism having an Nfa-1 protein ascompared to a control. The therapeutically effective amount will varydepending on the exact chemical structure of the Nfa-1 inhibitor, theexact organism having an Nfa-1 protein causing the infection in thesubject, the severity and/or type of the infection or other disease,disorder, syndrome, or symptom thereof being treated, the route ofadministration, the time of administration, the rate of excretion, thedrug combination, the judgment of the treating physician, the dosageform, and the age, weight, general health, sex and/or diet of thesubject to be treated.

As used herein, “synergistic effect,” “synergism,” or “synergy” refersto an effect arising between two or more molecules, compounds,substances, factors, or compositions that is greater than or differentfrom the sum of their individual effects.

As used herein, “additive effect” refers to an effect arising betweentwo or more molecules, compounds, substances, factors, or compositionsthat is equal to or the same as the sum of their individual effects.

As used herein, “treat,” “treating,” “treatment” and grammaticalvariations thereof as used herein include partially or completelydelaying, alleviating, mitigating or reducing the intensity of one ormore attendant symptoms of a disorder or condition such as an atypicalprotein kinase C enzyme abnormality and/or alleviating, mitigating orimpeding one or more causes of a disorder or condition such as anatypical protein kinase C enzyme abnormality. Treatments according tothe embodiments disclosed herein may be applied preventively,prophylactically, pallatively or remedially. In some instances, theterms “treat,” “treating,” “treatment” and grammatical variationsthereof include partially or completely reducing a condition or symptomassociated with, for example, an atypical protein kinase C enzymeabnormality as compared with prior to treatment of the subject or ascompared with the incidence of such condition or symptom in a general orstudy population.

As used herein, “identity,” is a relationship between two or morepolypeptide sequences, as determined by comparing the sequences. In theart, “identity” also refers to the degree of sequence relatednessbetween polypeptide as determined by the match between strings of suchsequences. “Identity” can be readily calculated by known methods,including, but not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., Ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity are codified in publicly availablecomputer programs. The percent identity between two sequences can bedetermined by using analysis software (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, Madison Wis.) that incorporatesthe Needelman and Wunsch (J. Mol. Biol., 1970, 48: 443-453) algorithm(e.g., NBLAST, and XBLAST). The default parameters are used to determinethe identity for the polypeptides of the present disclosure.

As used herein, “structural analogue” refers to a compound, molecule,protein, and the like, that has a structure similar to that of anothercompound, but is different in one aspect, such as an atom or functionalgroup.

As used herein, “functional analogue” refers to a compound molecule,nucleotide sequence, protein, and the like, that has the same or similarphysical, chemical, biochemical, pharmacological propertieis, or elicitthe same effect as another molecule, protein, and the like. In someembodiments, functional analogues are also structural analogues. Inother embodiments, functional analogues are not structural analogues.

As used herein, “analogue” refers to both the terms “structuralanalogue” and “functional analogue.”

As used herein, “heterologue” refers to compounds, molecules, nucleotidesequences (including genes), and polypeptide sequences (includingpeptides and proteins) that are different in both activity (function)and sequence or chemical structure.

As used herein, “homologue” refers to a polypeptide sequence that sharesa threshold level of similarly and/or identity as determined byalignment of matching amino acids. Two or more polypeptides determinedto be homologues are said to be homologues. Homology is a qualitativeterm that describes the relationship between polypeptide sequences thatis based upon the quantitative similarity.

As used herein, “paralog” refers to a homologue produced via geneduplication of a gene. In other words, paralogs are homologues thatresult form divergent evolution from a common ancesteral gene.

As used herein, “orthologs” refers to homologues produced by speciationfollowed by divergence of sequence but not activity in separate species.When speciation follows duplication and one homologue sorts with onespecies and the other copy sorts with the other species, subsequentdivergence of the duplicated sequence is associated with one or theother species. Such species specific homologues are referred to hereinas orthologs.

As used herein, “xenologs” are homologues resulting from horizontal genetransfer.

As used herein, “similarity” is a quantitative term that defines thedegree of sequence match between two compared polypeptide sequences.

As used herein, “Nfa-1 containing organism” and “organism having anNfa-1 protein” refers to an organism that contains or expresses an Nfa-1protein as defined herein.

As used herein “Nfa-1 protein” refers to and includes N. fowleri Nfa-1and any functional and structural analogues, homologues, paralogs,orthologs, xenologs of N. fowleri Nfa-1. “Nfa-1 protein” also includesany functional and structural analogues, homologues, paralogs,orthologs, xenologs of the Fe-oxygen binding site N. fowleri Nfa-1. Thethreshold for considering a protein an N. fowleri Nfa-1 homologue is anidentity of 25% or higher and a similarity of 30% or higher at the aminoacid level.

Discussion

Pathogenic and opportunistic free-living amoebae, such as Acanthanoebassp. Balamuthia mandrillaris, Sappinia diploidea, and Naegleria fowleri,are protists that occur world-wide and can cause serious and sometimesfatal infections in humans and other animals. Acanthanoeba ssp. and B.mandrillaris cause granulomatous amoebic encephalitis (GAE), which isinsidious, chronic, and mostly fatal. Acanthanoeba ssp. and B.mandrillaris most often cause disease in immunocompromised or otherwisedebilitated individuals. In addition to causing central nervous systeminfections, Acanthanoeba ssp. can cause vision-threatening keratitis.Both Acanthanoeba ssp. and B. mandrillaris can cause infection of thelungs and skin. S. diploidea, which is found in soil contaminated withfecal matter from elk, bison, and cattle, can cause encephalitis inhealthy individuals. N. fowleri causes an acute necrotizing andhemorrhagic meningoencephalitis (PAM), which results in death in over95% of cases.

While alarmingly devastating in nature, infections from free-livingamoebae are rare and typically affect fewer than 200,000 people. Giventhe rarity of pathogenic amoebae diseases, little research and productdevelopment has gone into developing treatment. As such, no effectiveand efficient therapy exists for the treatment of pathogenic andopportunistic free-living amoebae, such as those described above. Withthat said, described herein are compounds, pharmaceutical formulations,and methods of using the compounds and pharmaceutical formulations totreat or prevent amoebae infection or symptom thereof in a subjectinfected with or suspected of being infected with an amoebae or otherorganism having an Nfa-1 protein.

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Nfa-1 Inhibitors and Formulations Thereof

Nfa-1 Inhibitors

The pathology of some amoebic infections include the generation ofreactive oxygen species (ROS), which can contribute to the cytotoxicityobserved in some of these diseases. It has been known that cellsinfected with an amoeba (e.g. N. fowleri) have increased ROS. NADPHoxidase (NOX) of the host cell has been demonstrated to be involved inthe generation of ROS in cells infected with amoeba. However asdemonstrated herein, host-cell NOX is not the only source for ROS inamoebae infected cells.

Nfa-1 is a protein recently identified to be a virulence factor in N.fowleri. See Kang et. al. 2005. Clin. Diagn. Lab. Immunol 12:873-876 andJeong et al. 2005. Infect. Immun. 73:4098-4105. Nfa-1 belongs to theFe-binding hemerythrin protein family based on its amino acid sequence,but no biochemical characterization or enzymatic function has beenidentified. Until now, it was only known that Nfa-1 appears to play arole in the cell contact mechanism of N. fowleri (Id.), which wasthought to play a role in cytotoxic effect of the organism. As shown inthe Examples herein, amoebic Nfa-1 has NAD(P)H-dependent oxidoreductaseactivity and therefore can contribute to the production of ROS ininfected cells. Therefore, Nfa-1 can play a role in the cytotoxicityobserved in amoebic infections and other organisms having an Nfa-1protein.

The compositions described herein can alter Nfa-1 oxidoreductaseactivity. In some embodiments, the compounds described herein reduceand/or inhibit Nfa-1 oxidoreductase activity. In some embodiments, thecompositions described herein can reduce the amount of ROS in a subjector cell thereof infected with an organism having an Nfa-1 protein. Inother embodiments, the compositions described herein can reducecytotoxicity in a subject infected or suspected of being infected withan organism having an Nfa-1 protein. The compositions described hereincan be made using methods generally known in the art.

The compositions can have a formula according to Formula I:

where R₁ can be N or C, R₂ can be O or H, R₃ can be N or C, R₄ can be H,OH, O, or S, can be R₅ is N or C, R₆ can be N or C, R₇ can be N or C, R₈can be N or C, and R₉ can be H or O.

In other embodiments, the composition can have a formula according toany one of Formula II, Formula III, Formula IV, or Formula V:

In further embodiments, the compositions can be suitable derivatives ofany one of Formula I, Formula II, Formula III, Formula IV, or Formula V.Suitable derivatives can be derivatives that alter Nfa-1 oxidoreductaseactivity. In some embodiments, the suitable derivatives can reduce ROSin a subject or cell thereof infected with or suspected of beinginfected with an organism having an Nfa-1 protein. In other embodiments,the suitable derivatives can reduce cytotoxicity in a subject orinfected with or suspected of being infected with an organism having anNfa-1 protein.

The compositions can also have a formula according to Formula VI:

where R₁ can be H or OH and R₂ can be H, COCH₃, or COH.

In some embodiments the composition can have a formula according toFormula VII, Formula VIII, Formula IX, or Formula X.

In further embodiments, the compositions can be suitable derivatives ofany one of Formula VI, Formula VII, Formula VIII, Formula IX, or FormulaX. Suitable derivatives can be derivatives that alter Nfa-1oxidoreductase activity. In some embodiments, the suitable derivativescan reduce ROS in a subject or cell thereof infected with or suspectedof being infected with an organism having an Nfa-1 protein. In otherembodiments, the suitable derivatives can reduce cytotoxicity in asubject or infected with or suspected of being infected with an organismhaving an Nfa-1 protein.

Pharmaceutical Formulations

The Nfa-1 inhibitors described herein can be provided to a subject aloneor as an ingredient, such as an active ingredient, in a pharmaceuticalformulation. As such, also described herein are pharmaceuticalformulations containing one or more of the Nfa-1 inhibitors describedherein. In some embodiments, the pharmaceutical formulations contain atherapeutically effective amount of one or more Nfa-1 inhibitorsdescribed herein.

The pharmaceutical formulations can be administered to a subject in needthereof. In some embodiments, the subject in need thereof can besuspected of being infected with an organism having an Nfa-1 protein. Insome embodiments, the organism can be N. fowleri, N. gruberi,Acanthamoeba (“A.”) hatchetti, A. healyi, A. polyphaga, A. rhysodes, A.astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola. In other embodiments the subjectin need thereof can be infected with or be suspected of being infectedwith an organism having an Nfa-1 protein. In other embodiments, theNfa-1 inhibitors can be used in the manufacture of a medicament for thetreatment or prevention of an infection or symptom thereof caused by anorganism having an Nfa-1 protein.

Pharmaceutically Acceptable Carriers and Auxiliary Ingredients andAgents

The pharmaceutical formulations containing a therapeutically effectiveamount of a Nfa-1 inhibitor described herein can further include apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, but are not limited to, water, saltsolutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinylpyrrolidone, which do not deleteriously react with the activecomposition.

The pharmaceutical formulations can be sterilized, and if desired, mixedwith auxiliary agents, such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances, and the likewhich do not deleteriously react with the active composition.

In addition to the therapeutically effective amount of the Nfa-1inhibitors, the pharmaceutical formulation can also include an effectiveamount of auxiliary active agents, including but not limited to, DNA,RNA, amino acids, peptides, polypeptides, antibodies, aptamers,ribozymes, guide sequences for ribozymes that inhibit translation ortranscription of essential tumor proteins and genes, hormones,immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics,antispasmodics, anti-inflammatories, anti-histamines, anti-infectives,and chemotherapeutics.

Suitable hormones include, but are not limited to, amino-acid derivedhormones (e.g. melatonin and thyroxine), small peptide hormones andprotein hormones (e.g. thyrotropin-releasing hormone, vasopressin,insulin, growth hormone, luteinizing hormone, follicle-stimulatinghormone, and thyroid-stimulating hormone), eiconsanoids (e.g.arachidonic acid, lipoxins, and prostaglandins), and steroid hormones(e.g. estradiol, testosterone, tetrahydro testosteron cortisol).

Suitable immunomodulators include, but are not limited to, prednisone,azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins(e.g. IL-2, IL-7, and IL-12), cytokines (e.g. interferons (e.g. IFN-α,IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulatingfactor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7),cytosine phosphate-guanosine, oligodeoxynucleotides, glucans,antibodies, and aptamers).

Suitable antipyretics include, but are not limited to, non-steroidalanti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide),aspirin and related salicylates (e.g. choline salicylate, magnesiumsalicylae, and sodium salicaylate), paracetamol/acetaminophen,metamizole, nabumetone, phenazone, and quinine.

Suitable anxiolytics include, but are not limited to, benzodiazepines(e.g. alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate,diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, andtofisopam), serotenergic antidepressants (e.g. selective serotoninreuptake inhibitors, tricyclic antidepresents, and monoamine oxidaseinhibitors), mebicar, afobazole, selank, bromantane, emoxypine,azapirones, barbiturates, hydroxyzine, pregabalin, validol, and betablockers.

Suitable antipsychotics include, but are not limited to, benperidol,bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone,fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine,cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine,perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine,promazine, promethazine, prothipendyl, thioproperazine, thioridazine,trifluoperazine, triflupromazine, chlorprothixene, clopenthixol,flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine,prothipendyl, carpipramine, clocapramine, molindone, mosapramine,sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine,clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride,olanzaprine, paliperidone, perospirone, quetiapine, remoxipride,risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie,befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine,pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, andzicronapine.

Suitable analgesics include, but are not limited to,paracetamol/acetaminophen, non-steroidal anti-inflammants (e.g.ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g.rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine,oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine),tramadol, norepinephrine, flupiretine, nefopam, orphenadrine,pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone,ketobemidone, piritramide, and aspirin and related salicylates (e.g.choline salicylate, magnesium salicylate, and sodium salicylate).

Suitable antispasmodics include, but are not limited to, mebeverine,papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine,metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene,baclofen, tizanidine, and dantrolene.

Suitable anti-inflammatories include, but are not limited to,prednisone, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen,ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib,celecoxib, and etoricoxib), and immune selective anti-inflammatoryderivatives (e.g. submandibular gland peptide-T and its derivatives).

Suitable anti-histamines include, but are not limited to, H₁-receptorantagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine,buclizine, bromodiphenhydramine, carbinoxamine, cetirizine,chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine,desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate,dimetindene, diphenhydramine, doxylamine, ebasine, embramine,fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine,mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine,phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine,tripelennamine, and triprolidine), H₂-receptor antagonists (e.g.cimetidine, famotidine, lafutidine, nizatidine, rafitidine, androxatidine), tritoqualine, catechin, cromoglicate, nedocromil, andβ2-adrenergic agonists.

Suitable anti-infectives include, but are not limited to, amebicides(e.g. nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine,miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g.paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin),anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel,abendazole, thiabendazole, oxamniquine), antifungals (e.g. azoleantifungals (e.g. itraconazole, fluconazole, posaconazole, ketoconazole,clotrimazole, miconazole, and voriconazole), echinocandins (e.g.caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine,flucytosine, and polyenes (e.g. nystatin, and amphotericin b),antimalarial agents (e.g. pyrimethamine/sulfadoxine,artemether/lumefantrine, atovaquone/proquanil, quinine,hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine,and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g.aminosalicylic acid), isoniazid/rifampin,isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethambutol,rifampin, rifabutin, rifapentine, capreomycin, and cycloserine),antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine,emtricitabine/tenofovir,cobicistat/elvitegravir/emtricitabine/tenofovir,efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine,lamivudine/zidovudine, emtricitabine/tenofovir,emtricitabine/opinavir/ritonavir/tenofovir, interferonalfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir,dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir,zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delaviridine,nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine,tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine,telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir,fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir,amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir,famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g.doripenem, meropenem, ertapenem, and cilastatin/imipenem),cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin,cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil,loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime,cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, andceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin,oritavancin, and telvancin), glycylcyclines (e.g. tigecycline),leprostatics (e.g. clofazimine and thalidomide), lincomycin andderivatives thereof (e.g. clindamycin and lincomycin), macrolides andderivatives thereof (e.g. telithromycin, fidaxomicin, erthromycin,azithromycin, clarithromycin, dirithromycin, and troleandomycin),linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol,fosfomycin, metronidazole, aztreonam, bacitracin, penicillins(amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin,ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam,piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procainepenicillin, oxaxillin, dicloxacillin, and nafcillin), quinolones (e.g.lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin,ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin,nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin,and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim,sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline,demeclocycline, minocycline, doxycycline/salicyclic acid,doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), andurinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin,cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

Suitable chemotherapeutics include, but are not limited to, paclitaxel,brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus,pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine,ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin,bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide,lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin,ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine,dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide,epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab,vismodegib, asparginase Erwinia chrysanthemi, amifostine, etoposide,flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate,methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinibmesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan,ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferonalfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel,ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim,cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib,mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan,gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim,sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone,pegaspargase, denileukin diftitox, alitretinoin, carboplatin,pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin,mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide,dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine,thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene,temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustinehydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin,panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib,teniposide, leucovorin, crizotinib, capecitabine, enzalutamide,ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone,epothilone, tafluposide, azathioprine, doxifluridine, vindesine, andall-trans retinoic acid.

Effective Amounts of the Nfa-1 Inhibitors and Auxiliary Agents

The pharmaceutical formulations can contain a therapeutically effectiveamount of an Nfa-1 inhibitor and/or a therapeutically effective amountof an auxiliary agent. In some embodiments, the therapeuticallyeffective amount of the Nfa-1 inhibitor can range from about 0.01 mg/kgto about 20 mg/kg. In some embodiments, the therapeutically effectivemount of the Nfa-1 inhibitor can range from about 1 mg/kg to about 6mg/kg.

In embodiments where there is an auxiliary active agent contained in thepharmaceutical formulation in addition to the Nfa-1 inhibitor, thetherapeutically effective amount of the auxiliary active agent will varydepending on the auxiliary active agent. In some embodiments, theeffective amount of the auxiliary active agent ranges from 0.001micrograms to about 1 milligrams. In other embodiments, the effectiveamount of the auxiliary active agent ranges from about 0.01 IU to about1000 IU. In further embodiments, the effective amount of the auxiliaryactive agent ranges from 0.001 mL to about 1 mL. In yet otherembodiments, the effective amount of the auxiliary active agent rangesfrom about 1% w/w to about 50% w/w of the total pharmaceuticalformulation. In additional embodiments, the effective amount of theauxiliary active agent ranges from about 1% v/v to about 50% v/v of thetotal pharmaceutical formulation. In still other embodiments, theeffective amount of the auxiliary active agent ranges from about 1% w/vto about 50% w/v of the total pharmaceutical formulation.

The auxiliary active agent can be included in the pharmaceuticalformulation or can exist as a stand-alone compound or pharmaceuticalformulation that is administered contemporaneously or sequentially withthe conjugate compound, derivative thereof or pharmaceutical formulationthereof. In embodiments where the auxiliary active agent is astand-alone compound or pharmaceutical formulation, the therapeuticallyeffective amount of the auxiliary active agent can vary depending on theauxiliary active agent used. In some of these embodiments, thetherapeutically effective amount of the auxiliary active agent can rangefrom 0.001 micrograms to about 1000 grams. In other embodiments, thetherapeutically effective amount of the auxiliary active agent can rangefrom about 0.01 IU to about 1000 IU. In further embodiments, thetherapeutically effective amount of the auxiliary active agent can rangefrom 0.001 mL to about 1 mL. In yet other embodiments, thetherapeutically effective amount of the auxiliary active agent can rangefrom about 1% w/w to about 50% w/w of the total auxiliary active agentpharmaceutical formulation. In additional embodiments, thetherapeutically effective amount of the auxiliary active agent can rangefrom about 1% v/v to about 50% v/v of the total pharmaceuticalformulation. In still other embodiments, the therapeutically effectiveamount of the auxiliary active agent can range from about 1% w/v toabout 50% w/v of the total auxiliary agent pharmaceutical formulation.

Dosage Forms

In some embodiments, the pharmaceutical formulations described hereinmay be in a dosage form. The dosage forms can be adapted foradministration by any appropriate route. Appropriate routes include, butare not limited to, oral (including buccal or sublingual), rectal,intraocular, inhaled, intranasal, topical (including buccal, sublingual,or transdermal), vaginal, intraurethral, parenteral, intracranial,subcutaneous, intramuscular, intravenous, and intradermal. Suchformulations may be prepared by any method known in the art.

Dosage forms adapted for oral administration can be discrete dosageunits such as capsules, pellets or tablets, powders or granules,solutions, or suspensions in aqueous or non-aqueous liquids; ediblefoams or whips, or in oil-in-water liquid emulsions or water-in-oilliquid emulsions. In some embodiments, the pharmaceutical formulationsadapted for oral administration also include one or more agents whichflavor, preserve, color, or help disperse the pharmaceuticalformulation. Dosage forms prepared for oral administration can also bein the form of a liquid solution that can be delivered as a foam, spray,or liquid solution. In some embodiments, the oral dosage form cancontain about 100 mg, about 200 mg, or about 300 mg of a pharmaceuticalformulation containing a therapeutically effective amount or anappropriate fraction thereof of an Nfa-1 inhibitor. The oral dosage formcan be administered to a subject in need thereof. In some embodiments,this is a subject infected with or suspected of being infected with anorganism having an Nfa-1 protein.

Where appropriate, the dosage forms described herein can bemicroencapsulated. The dosage form can also be prepared to prolong orsustain the release of any ingredient. In some embodiments, the Nfa-1inhibitor is the ingredient whose release is delayed. In otherembodiments, the release of an auxiliary ingredient is delayed. Suitablemethods for delaying the release of an ingredient include, but are notlimited to, coating or embedding the ingredients in material inpolymers, wax, gels, and the like. Delayed release dosage formulationscan be prepared as described in standard references such as“Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York,Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). Thesereferences provide information on excipients, materials, equipment, andprocesses for preparing tablets and capsules and delayed release dosageforms of tablets and pellets, capsules, and granules. The delayedrelease can be anywhere from about an hour to about 3 months or more.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and hydroxypropyl methylcellulose acetate succinate;polyvinyl acetate phthalate, acrylic acid polymers and copolymers, andmethacrylic resins that are commercially available under the trade nameEUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, andpolysaccharides.

Coatings may be formed with a different ratio of water soluble polymer,water insoluble polymers, and/or pH dependent polymers, with or withoutwater insoluble/water soluble non polymeric excipient, to produce thedesired release profile. The coating is either performed on the dosageform (matrix or simple) which includes, but is not limited to, tablets(compressed with or without coated beads), capsules (with or withoutcoated beads), beads, particle compositions, “ingredient as is”formulated as, but not limited to, suspension form or as a sprinkledosage form.

Dosage forms adapted for topical administration can be formulated asointments, creams, suspensions, lotions, powders, solutions, pastes,gels, sprays, aerosols, or oils. In some embodiments for treatments ofthe eye or other external tissues, for example the mouth or the skin,the pharmaceutical formulations are applied as a topical ointment orcream. When formulated in an ointment, the Nfa-1 inhibitor, auxiliaryactive ingredient, and/or pharmaceutically acceptable salt thereof canbe formulated with a paraffinic or water-miscible ointment base. Inother embodiments, the active ingredient can be formulated in a creamwith an oil-in-water cream base or a water-in-oil base. Dosage formsadapted for topical administration in the mouth include lozenges,pastilles, and mouth washes.

Dosage forms adapted for nasal or inhalation administration includeaerosols, solutions, suspension drops, gels, or dry powders. In someembodiments, the Nfa-1 inhibitor, derivative thereof, auxiliary activeingredient, and/or pharmaceutically acceptable salt thereof in a dosageform adapted for inhalation is in a particle-size-reduced form that isobtained or obtainable by micronization. In some embodiments, theparticle size of the size reduced (e.g. micronized) compound or salt orsolvate thereof, is defined by a D50 value of about 0.5 to about 10microns as measured by an appropriate method known in the art. Dosageforms adapted for administration by inhalation also include particledusts or mists. Suitable dosage forms wherein the carrier or excipientis a liquid for administration as a nasal spray or drops include aqueousor oil solutions/suspensions of an active ingredient, which may begenerated by various types of metered dose pressurized aerosols,nebulizers, or insufflators.

In some embodiments, the dosage forms are aerosol formulations suitablefor administration by inhalation. In some of these embodiments, theaerosol formulation contains a solution or fine suspension of a Nfa-1inhibitor, auxiliary active ingredient, and/or pharmaceuticallyacceptable salt thereof a pharmaceutically acceptable aqueous ornon-aqueous solvent. Aerosol formulations can be presented in single ormulti-dose quantities in sterile form in a sealed container. For some ofthese embodiments, the sealed container is a single dose or multi-dosenasal or an aerosol dispenser fitted with a metering valve (e.g. metereddose inhaler), which is intended for disposal once the contents of thecontainer have been exhausted.

Where the aerosol dosage form is contained in an aerosol dispenser, thedispenser contains a suitable propellant under pressure, such ascompressed air, carbon dioxide, or an organic propellant, including butnot limited to a hydrofluorocarbon. The aerosol formulation dosage formsin other embodiments are contained in a pump-atomizer. The pressurizedaerosol formulation can also contain a solution or a suspension of aconjugate compound, derivative thereof, auxiliary active ingredient,and/or pharmaceutically acceptable salt thereof. In further embodiments,the aerosol formulation also contains co-solvents and/or modifiersincorporated to improve, for example, the stability and/or taste and/orfine particle mass characteristics (amount and/or profile) of theformulation. Administration of the aerosol formulation can be once dailyor several times daily, for example 2, 3, 4, or 8 times daily, in which1, 2, or 3 doses are delivered each time.

For some dosage forms suitable and/or adapted for inhaledadministration, the pharmaceutical formulation is a dry powder inhalableformulations. In addition to the Nfa-1 inhibitor, auxiliary activeingredient, and/or pharmaceutically acceptable salt thereof, such adosage form can contain a powder base such as lactose, glucose,trehalose, manitol, and/or starch. In some of these embodiments, theconjugate compund, derivative thereof, auxiliary active ingredient,and/or pharmaceutically acceptable salt thereof is in a particle-sizereduced form. In further embodiments, a performance modifier, such asL-leucine or another amino acid, cellobiose octaacetate, and/or metalssalts of stearic acid, such as magnesium or calcium stearate.

In some embodiments, the aerosol formulations are arranged so that eachmetered dose of aerosol contains a predetermined amount of an activeingredient, such as the one or more of the compounds described herein.

Dosage forms adapted for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulations.Dosage forms adapted for rectal administration include suppositories orenemas.

Dosage forms adapted for parenteral administration and/or adapted forinjection can include aqueous and/or non-aqueous sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats,solutes that render the composition isotonic with the blood of thesubject, and aqueous and non-aqueous sterile suspensions, which caninclude suspending agents and thickening agents. The dosage formsadapted for parenteral administration can be presented in a single-unitdose or multi-unit dose containers, including but not limited to sealedampoules or vials. The doses can be lyophilized and resuspended in asterile carrier to reconstitute the dose prior to administration.Extemporaneous injection solutions and suspensions can be prepared insome embodiments, from sterile powders, granules, and tablets.

Dosage forms adapted for ocular administration can include aqueousand/or non-aqueous sterile solutions that can optionally be adapted forinjection, and which can optionally contain anti-oxidants, buffers,bacteriostats, solutes that render the composition isotonic with the eyeor fluid contained therein or around the eye of the subject, and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

For some embodiments, the dosage form contains a predetermined amount ofan Nfa-1 inhibitor per unit dose. In an embodiment, the predeterminedamount of the Nfa-1 inhibitor is a therapeutically effective amount ofthe Nfa-1 inhibitor to treat, prevent, or mitigate the symptoms of aninfection with an organism having an Nfa-1 protein. In otherembodiments, the predetermined amount of the Nfa-1 inhibitor is anappropriate fraction of the effective amount of the active ingredient.Such unit doses may therefore be administered once or more than once aday. Such pharmaceutical formulations may be prepared by any of themethods well known in the art.

Methods of Using the Nfa-1 Inhibitors and Formulations Thereof

The Nfa-1 Inhibitors and pharmaceutical formulations thereof describedherein can be used for treatment or prevention of a disease, disorder,syndrome, or a symptom thereof. In some embodiments, the Nfa-1 Inhibitorcan be used to treat a subject infected with or suspected of beinginfected with an organism having an Nfa-1 protein. In some embodiments,the organism having an Nfa-1 protein can be N. fowleri, N. gruberi,Acanthamoeba (“A.”) hatchetti, A. healyi, A. polyphaga, A. rhysodes, A.astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola.

An amount of the Nfa-1 inhibitor and pharmaceutical formulationsthereof, described herein can be administered to a subject in needthereof one or more times per day, week, month, or year. In someembodiments, the subject has one or more symptoms of a disease,condition, or syndrome. In some embodiments, the Nfa-1 Inhibitor can beused to treat a subject infected with or suspected of being infectedwith an organism having an Nfa-1 protein. In some embodiments, theorganism having an Nfa-1 protein can be N. fowleri, N. gruberi,Acanthamoeba hatchetti, A. healyi, A. polyphaga, A. rhysodes, A.astronyxis, A. divionensis, A. castellanii, Entamoeba histolytica, E.invadens, E. dispar, Balamuthia mandrillaris, Sappinia diploidea,Pyrococcus furiosus, Clostridium acetobutylicum, Desulfovibrio vulgaris,Burkholderia pseudomallei, Desulfurococcus mucosus, Methanococcusjannaschii, Riftia pachyptila, Phascolopsis gouldii, Periserrulaleucophryna, Perinereis aibuhitensis, Theromyzon tessulatum, Hirudomedicinalis, or Themiste zostericola

In some embodiments, the amount administered can be the therapeuticallyeffective amount of the Nfa-1 inhibitor or pharmaceutical formulationsthereof. For example, the Nfa-1 inhibitor or pharmaceutical formulationsthereof can be administered in a daily dose. This amount may be given ina single dose per day. In other embodiments, the daily dose may beadministered over multiple doses per day, in which each containing afraction of the total daily dose to be administered (sub-doses). In someembodiments, the amount of doses delivered per day is 2, 3, 4, 5, or 6.In further embodiments, the compounds, formulations, or salts thereofare administered one or more times per week, such as 1, 2, 3, 4, 5, or 6times per week. In other embodiments, the Nfa-1 inhibitors orpharmaceutical formulations thereof are administered one or more timesper month, such as 1 to 5 times per month. In still further embodiments,the Nfa-1 inhibitor or pharmaceutical formulations thereof areadministered one or more times per year, such as 1 to 11 times per year.

The Nfa-1 inhibitor or pharmaceutical formulations thereof can beco-administered with a secondary agent by any convenient route. Thesecondary agent is a separate compound and/or formulation from the Nfa-1inhibitor or pharmaceutical formulation thereof. The secondary agent canbe administered simultaneously with the Nfa-1 inhibitor orpharmaceutical formulation thereof or sequentially with the Nfa-1inhibitor or pharmaceutical formulation thereof. The secondary agent canhave an additive or synergistic effect to the Nfa-1 inhibitor orpharmaceutical formulation thereof. Suitable secondary agents include,but are not limited to, DNA, RNA, amino acids, peptides, polypeptides,antibodies, aptamers, ribozymes, guide sequences for ribozymes thatinhibit translation or transcription of essential tumor proteins andgenes, hormones, immunomodulators, antipyretics, anxiolytics,antipsychotics, analgesics, antispasmodics, anti-inflammatories,anti-histamines, anti-infectives, and chemotherapeutics.

Suitable hormones include, but are not limited to, amino-acid derivedhormones (e.g. melatonin and thyroxine), small peptide hormones andprotein hormones (e.g. thyrotropin-releasing hormone, vasopressin,insulin, growth hormone, luteinizing hormone, follicle-stimulatinghormone, and thyroid-stimulating hormone), eiconsanoids (e.g.arachidonic acid, lipoxins, and prostaglandins), and steroid hormones(e.g. estradiol, testosterone, tetrahydro testosteron cortisol).

Suitable immunomodulators include, but are not limited to, prednisone,azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins(e.g. IL-2, IL-7, and IL-12), cytokines (e.g. interferons (e.g. IFN-α,IFN-β, IFN-ε, IFN-78 , IFN-ω, and IFN-γ), granulocyte colony-stimulatingfactor, and imiquimod), chemokines (e.g. CCL3, CCL26 and CXCL7),cytosine phosphate-guanosine, oligodeoxynucleotides, glucans,antibodies, and aptamers).

Suitable antipyretics include, but are not limited to, non-steroidalanti-inflammants (e.g. ibuprofen, naproxen, ketoprofen, and nimesulide),aspirin and related salicylates (e.g. choline salicylate, magnesiumsalicylae, and sodium salicaylate), paracetamol/acetaminophen,metamizole, nabumetone, phenazone, and quinine.

Suitable anxiolytics include, but are not limited to, benzodiazepines(e.g. alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate,diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, andtofisopam), serotenergic antidepressants (e.g. selective serotoninreuptake inhibitors, tricyclic antidepresents, and monoamine oxidaseinhibitors), mebicar, afobazole, selank, bromantane, emoxypine,azapirones, barbituates, hyxdroxyzine, pregabalin, validol, and betablockers.

Suitable antipsychotics include, but are not limited to, benperidol,bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone,fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine,cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine,perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine,promazine, promethazine, prothipendyl, thioproperazine, thioridazine,trifluoperazine, triflupromazine, chlorprothixene, clopenthixol,flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine,prothipendyl, carpipramine, clocapramine, molindone, mosapramine,sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine,clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride,olanzaprine, paliperidone, perospirone, quetiapine, remoxipride,risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie,befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine,pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, andzicronapine.

Suitable analgesics include, but are not limited to,paracetamol/acetaminophen, non-steroidal anti-inflammants (e.g.ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g.rofecoxib, celecoxib, and etoricoxib), opioids (e.g. morphine, codeine,oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine),tramadol, norepinephrine, flupiretine, nefopam, orphenadrine,pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone,ketobemidone, piritramide, and aspirin and related salicylates (e.g.choline salicylate, magnesium salicylae, and sodium salicaylate).

Suitable antispasmodics include, but are not limited to, mebeverine,papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine,metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene,baclofen, tizanidine, and dantrolene.

Suitable anti-inflammatories include, but are not limited to,prednisone, non-steroidal anti-inflammants (e.g. ibuprofen, naproxen,ketoprofen, and nimesulide), COX-2 inhibitors (e.g. rofecoxib,celecoxib, and etoricoxib), and immune selective anti-inflammatoryderivatives (e.g. submandibular gland peptide-T and its derivatives).

Suitable anti-histamines include, but are not limited to, H₁-receptorantagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine,buclizine, bromodiphenhydramine, carbinoxamine, cetirizine,chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine,desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate,dimetindene, diphenhydramine, doxylamine, ebasine, embramine,fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine,mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine,phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine,tripelennamine, and triprolidine), H₂-receptor antagonists (e.g.cimetidine, famotidine, lafutidine, nizatidine, rafitidine, androxatidine), tritoqualine, catechin, cromoglicate, nedocromil, andβ2-adrenergic agonists.

Suitable anti-infectives include, but are not limited to, amebicides(e.g. nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine,miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g.paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin),anthelmintics (e.g. pyrantel, mebendazole, ivermectin, praziquantel,abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g.azole antifungals (e.g. itraconazole, fluconazole, posaconazole,ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins(e.g. caspofungin, anidulafungin, and micafungin), griseofulvin,terbinafine, flucytosine, and polyenes (e.g. nystatin, and amphotericinb), antimalarial agents (e.g. pyrimethamine/sulfadoxine,artemether/lumefantrine, atovaquone/proquanil, quinine,hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine,and halofantrine), antituberculosis agents (e.g. aminosalicylates (e.g.aminosalicylic acid), isoniazid/rifampin,isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethanmbutol,rifampin, rifabutin, rifapentine, capreomycin, and cycloserine),antivirals (e.g. amantadine, rimantadine, abacavir/lamivudine,emtricitabine/tenofovir,cobicistat/elvitegravir/emtricitabine/tenofovir,efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine,lamivudine/zidovudine, emtricitabine/tenofovir,emtricitabine/opinavir/ritonavir/tenofovir, interferonalfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir,dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir,zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delaviridine,nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine,tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine,telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir,fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir,amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir,famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g.doripenem, meropenem, ertapenem, and cilastatin/imipenem),cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin,cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil,loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime,cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, andceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin,oritavancin, and telvancin), glycylcyclines (e.g. tigecycline),leprostatics (e.g. clofazimine and thalidomide), lincomycin andderivatives thereof (e.g. clindamycin and lincomycin), macrolides andderivatives thereof (e.g. telithromycin, fidaxomicin, erthromycin,azithromycin, clarithromycin, dirithromycin, and troleandomycin),linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol,fosfomycin, metronidazole, aztreonam, bacitracin, penicillins(amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin,ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam,piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procainepenicillin, oxaxillin, dicloxacillin, and nafcillin), quinolones (e.g.lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin,ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin,nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin,and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim,sulfasalazine, and sulfasoxazole), tetracyclines (e.g. doxycycline,demeclocycline, minocycline, doxycycline/salicyclic acid,doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), andurinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin,cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

Suitable chemotherapeutics include, but are not limited to, paclitaxel,brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus,pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine,ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin,bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide,lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin,ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine,dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide,epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab,vismodegib, asparginase Erwinia chrysanthemi, amifostine, etoposide,flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate,methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinibmesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan,ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferonalfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel,ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim,cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib,mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan,gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim,sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone,pegaspargase, denileukin diftitox, alitretinoin, carboplatin,pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin,mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide,dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine,thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene,temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustinehydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin,panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib,teniposide, leucovorin, crizotinib, capecitabine, enzalutamide,ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone,epothilone, tafluposide, azathioprine, doxifluridine, vindesine, andall-trans retinoic acid.

In embodiments where the Nfa-1 inhibitor or pharmaceutical formulationthereof is simultaneous co-administered with a secondary agent, theNfa-1 inhibitor or pharmaceutical formulation thereof is administered tothe subject at substantially the same time as the secondary agent. Asused in this context “substantially the same time” refers toadministration of the Nfa-1 inhibitor or pharmaceutical formulationthereof and a secondary agent where the period of time betweenadministration of the Nfa-1 inhibitor or pharmaceutical formulationthereof and a secondary agent is between 0 and 10 minutes.

In embodiments where the Nfa-1 inhibitor or pharmaceutical formulationthereof are sequentially co-administered with a secondary agent, theNfa-1 inhibitor or pharmaceutical formulation thereof can beadministered first followed by administration of the secondary agentafter a period of time. In other embodiments where the Nfa-1 inhibitoror pharmaceutical formulation thereof are sequentially co-administeredwith a secondary agent, the secondary agent can be administered firstfollowed by administration of the Nfa-1 inhibitor or pharmaceuticalformulation thereof after a period of time. In any embodiment, theperiod of time can range from 10 minutes to about 96 hours. In someembodiments the period of time can be about 10 minutes, about 30minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours,about 8 hours, about 10 hours, or about 12 hours. The sequentialadministration can be repeated as necessary over the course of theperiod of treatment.

The amount of the Nfa-1 inhibitors, pharmaceuticals formulationsthereof, and secondary agents described herein can be administered in anamount ranging from about 0.01 mg to about 10 g per day, as calculatedas the free or unsalted compounds or pharmaceutical formulations. Theamount of Nfa-1 inhibitors, pharmaceuticals formulations thereof, andsecondary agents described herein can be administered in an amountranging from about 0.01 μM to about 10 μM per day.

Kits Containing the Nfa-1 Inhibitors and Formulations Thereof

The Nfa-1 inhibitors and pharmaceuticals formulations thereof describedherein can be presented as a combination kit. As used herein, the terms“combination kit” or “kit of parts” refers to the Nfa-1 inhibitors orpharmaceuticals formulations thereof described herein and additionalcomponents that are used to package, sell, market, deliver, and/oradminister the combination of elements or a single element, such as theactive ingredient, contained therein. Such additional componentsinclude, but are not limited to, packaging, syringes, blister packages,bottles, and the like. When one or more of the components (e.g. activeagents) contained in the kit are administered simultaneously, thecombination kit can contain the active agents in a single pharmaceuticalformulation (e.g. a tablet) or in separate pharmaceutical formulations.

The combination kit can contain each agent, compound, pharmaceuticalformulation or component thereof, in separate compositions orpharmaceutical formulations. The separate compositions or pharmaceuticalformulations can be contained in a single package or in separatepackages within the kit. Also provided in some embodiments, are buffers,diluents, solubilization reagents, cell culture media and otherreagents. These additional components can be contained in a singlepackage or in separate packages within the kit.

In some embodiments, the combination kit also includes instructionsprinted on or otherwise contained in a tangible medium of expression.The instructions can provide information regarding the content of theNfa-1 inhibitors or pharmaceuticals formulations thereof, pharmaceuticalformulations thereof, and/or other auxiliary agent contained therein,safety information regarding the content of the Nfa-1 inhibitors,pharmaceuticals formulations thereof, pharmaceutical formulationsthereof, auxiliary agent, or secondary contained therein, informationregarding the dosages, indications for use, and/or recommended treatmentregimen(s) for the Nfa-1 inhibitors, pharmaceutical formulationsthereof, and/or other auxiliary or secondary agent contained therein. Insome embodiments, the instructions provide directions for administeringthe compounds, pharmaceutical formulations, or salts thereof to asubject infected with or suspected of being infected with an organismhaving an Nfa-1 protein. In some embodiments, organism having an Nfa-1protein can be N. fowleri, N. gruberi, Acanthamoeba hatchetti, A.healyi, A. polyphaga, A. rhysodes, A. astronyxis, A. divionensis, A.castellanii, Entamoeba histolytica, E. invadens, E. dispar, Balamuthiamandrillaris, Sappinia diploidea, Pyrococcus furiosus, Clostridiumacetobutylicum, Desulfovibrio vulgaris, Burkholderia pseudomallei,Desulfurococcus mucosus, Methanococcus jannaschii, Riftia pachyptila,Phascolopsis gouldii, Periserrula leucophryna, Perinereis aibuhitensis,Theromyzon tessulatum, Hirudo medicinalis, or Themiste zostericola.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1 Phylogram Analysis of Nfa-1 Proteins

Amino acid sequences of hemerythrin-related proteins from variousamoebae, bacteria, archaea, and polychaete worms with sequenced genomeswere obtained from NCBI GenBank or AmoebaDB online databases. Alignmentof amino acid sequences was completed using ClustalW2 and results weredisplayed as phylograms, which graphically demonstrates the identityrelationship between the amino acid sequences observed. Representativephylograms are shown in FIGS. 1 and 2. As demonstrated in FIG. 1, N.fowleri Nfa-1 hemerythrin (Hr) had the closest homology to N. gruberihemerythrins, followed by the Glade of bacterial and archaea Hr-relatedproteins.

Example 2 LIGPLOT Analysis of N. fowleri Nfa-1 Fe-Oxygen Protein-LigandInteractions

LIGPLOT of N. fowleri Nfa-1 bound with two ferric (Fe) ions and oxygenwas generated from the LIGPLOT and crystal structure of P. gouldii Hr asprovided in the online Protein Data Bank, PDBsum (PDBsum identification:“li4y” at 1.8 Å) (FIG. 3) following pairwise alignment of the N. fowleriNfa-1 with the P. gouldii Hr amino acid sequence by pairwise BLAST(NCBI/BLAST/blastp suite), which allowed identification of the specificamino acids in N. fowleri Nfa-1 that correspond to the specific aminoacids given in the P. gouldii Hr LIGPLOT in PDBsum. Hence the Hr-Febinding site was observed to be composed of five histidine (His)residues and two acidic residues (glutamate, Glu60 and aspartate,Asp112) that form ionic bonds (in blue) with two Fe ions. PhenylalaninePhe57 forms hydrophobic interactions (depicted as eyelashes) thatstabilize the Fe binding pocket. Hydrogen bonds and their apparentlengths in Angstrom (Å) are given in red (FIG. 3).

Example 3 Blast Search and Ligplot Analysis of Acanthamoeba Protein withNfa-1 Like Hemerythrin Domain

A BLAST search of the entire 119-amino acid N. fowleri Nfa-1 proteinsequence against Acanthamoeba genes in GenBank was conducted. The BLASTsearch revealed an Acanthamoeba castellanii protein (XP_004356500.1)with an Nfa-1 hemerythrin-like domain that showed 32% (37/117) identityand 52% (61/117) similarity at the amino acid level with the whole N.fowleri homologue (FIG. 5). The LIGPLOT (FIG. 4) and BLAST (FIG. 5)analysis of the Acanthamoeba hemerythrin revealed 100% amino acididentity for the Fe-oxygen binding site of the enzyme with the N.fowleri Nfa-1 oxidase as highlighted and marked with an asterik (*) inFIG. 5 for the eight respective amino acids that form the binding site.This suggests that the putative (Gen Bank Accession No. XP_004356500.1)protein is an Nfa-1 homologue.

Example 4 Molecular Structure of N. fowleri Nfa-1 Hemerythrin

FIGS. 6A and 6B demonstrate a side view (FIG. 6A) and top view (FIG. 6B)of a ribbon diagram of N. fowleri Nfa-1 Hr. The predicted proteinstructure of Nfa-1 was modeled in silico using Phascolopsis gouldii Hr(PDB: li4y 1.8 Å) as template (QMEAN4 score of 0.69). For in silicomodeling, amino acid sequences were pair-wise aligned with ClustalW2followed by protein modeling in SWISS-MODEL with the molecular structureshown as solid ribbon model (FIGS. 6A and 6B). The five histidine (His)residues of the Fe iron binding site are shown in yellow and the twoacidic residues glutamate Glu60 and aspartate Asp112 are marked in blue(FIGS. 6A and 6B). For clarity, only the ionic bonds between the fiveHis residues and two Fe ions are shown (FIGS. 6A and 6B).

FIGS. 12A-12B show the amino acid sequence (SEQ ID NO: 3) (FIG. 12A) andthe DNA sequence (SEQ ID NO: 4) (FIG. 12B for N. fowleri Nfa-1. TheGenBank Accession No. for SEQ ID NO: 3 is AAF35899 and the GenBankAccession No. for SEQ ID NO: 4 is AF230370. The eight underlined aminoacids (FIG. 12A) and nucleotides (FIG. 12B) correspond to the Nfa-1Fe-oxygen binding site as demonstrated in FIGS. 3, 5, and 6A-6B. Theseare His26, His56, Phe57, Glu60, His75, His79, His107, Asp112 in SEQ IDNO: 3, where the first Met is considered amino acid 1 in the sequence.The first Met in SEQ ID NO: 3 corresponds to the first three nucleotidesin SEQ ID NO: 4 (the “start” codon). The last three nucleotides (“TAA”)of SEQ ID NO: 4 correspond to the “stop” codon and thus do not have acorresponding amino acid in SEQ ID NO: 3. Nucleotides (nt)76-78,nt166-168, nt169-171, nt178-180, nt223-225, nt235-237, nt319-321,nt334-336 in SEQ ID NO: 4 correspond to the codons for the eight aminoacids that correspond to the Fe-oxygen binding site in SEQ ID NO: 3previously discussed.

Example 5 Nfa-1 Oxidoreductase Activity, Enzyme Kinetics, andPharmacological Inhibition Assay

Cloning of N. fowleri Nfa-1 Hemerythrin and Production of RecombinantNfa-1 Enzyme.

The N. fowleri Nfa-1 gene (360 bp, Gen Bank Accession No. AF230370) wasamplified by PCR from genomic N. fowleri DNA using PCR primers thatincluded the Nde1 and Sap1 restriction enzyme sites for subsequentcloning into the Nde1 and Sap1 sites of the bacterial expression vectorpTwin1 (forward primer with Nde1 site underlined; ATG start codon isindicated in bold:

SEQ ID NO: 1  5′-TAGCTACATATGGCCACTACTATTCCATCACCATTC-3′,reverse primer with Sap1 site underlined:

SEQ ID NO: 2  5′-CAGTATGCTCTTCTGCAAAGCACTCCCTTGTACTTCATATCAG-3′),which contained a thiol-inducible intein protease fused to achitin-binding domain. Recombinant Nfa-1 protein was produced in E. coliand purified by chitin-agarose chromatography followed by cleavage andelution of the purified Nfa-1 protein by 40 mM dithiothreitol (DTT) andsubsequent size-exclusion chromatography purification.

Measurement of Nfa-1 Hemerythrin Enzyme Kinetics and PharmacologicalInhibition Assays.

Enzymatic oxidase activity, kinetics and pharmacological drug inhibitionassays of recombinant N. fowleri Nfa-1 hemerythrin were determined invitro using NADH or NADPH as the substrate and electron donor andferricyanide as electron acceptor. Various drugs and inhibitors specificfor xanthine oxidase-like enzymes, NAD(P)H oxidases, or NADH peroxidasewere included in the pharmacological inhibition assays.

Initial-Rate Enzyme Kinetics of N. fowleri Nfa-1 Hemerythrin Using NADHor NADPH as Substrates.

Enzyme kinetics were determined using NADH or NADPH at 10-200 μMconcentration as electron donor and ferricyanide (200 μM) as electronacceptor for initial-rate enzyme kinetics of 40 sec at 23° C. ApparentK_(m) values of 64.5 μM (NADH) and 22.3 μM (NADPH), and apparent k_(cat)values of 4.4/sec (NADH) and 3.3/sec (NADPH) were determined byMichaelis-Menten kinetics analysis. Results of the Nfa-1 enzyme activityassays are demonstrated in FIGS. 7A-7B and FIG. 8.

Inhibitor Profile and Pharmacology of N. fowleri Nfa-1 Oxidase In Vitro.

Enzyme activity in the presence of various substrate analogues andinhibitors were determined, including the NADH substrate analoguehypoxanthine (K_(i)=1 mM), the xanthine oxidase inhibitor Allopurinol(K_(i)=134 μM), the NAD(P)H oxidase inhibitors DPI (diphenyleneiodoniumchloride) (K_(i)=19 μM) and Apocynin (K_(i)=17 μM), the NADH peroxidaseinhibitors AgNO₃ (K_(i)=345 μM) and pHMB (para-hydroxymercuribenzoate)(K_(i)=78 μM), and the NADH peroxidase activator H₂O₂. The results aredemonstrated in FIG. 9.

Example 6 Effect of Nfa-1 Inhibitors on N. fowleri-Induced Cytotoxicity

Naegleria fowleri strain Nf69 (ATCC 30215) was cultivated at about 33°C. in modified Nelson's medium (about 1 mM Na₂HPO₄, about 1 mM KH₂PO₄,about 2.05 mM NaCl, about 16.2 μM MgSO₄, about 27.2 μM CaCl₂, about 0.17g/L DIFCO Liver infusion broth, about 9.4 mM glucose) supplemented withabout 10% heat-inactivated fetal bovine serum. For cytotoxicity assays,Chinese hamster ovary (CHO) cells were grown at about 37° C. in DMEMmedium supplemented with about 10% fetal bovine serum until culturesreached about 70-80% confluency. Subsequently cultures were infectedwith N. fowleri cells at a ratio of 1 N. fowleri amoeba per 5-10 CHOcells and co-cultivated overnight for about 13 hours at about 37° C. Thecytotoxicity effect of N. fowleri amoebae on CHO cells was measured byan LDH-based cytotoxicity assay that quantifies the release of lactatedehydrogenase (LDH) from dead cells at 490 nm wavelength absorbance forcalculation of amoeba-induced cytotoxicity to the mammalian host cells.

Cytotoxicity of Naegleria fowleri amoebae was measured with CHO cellsco-cultured with N. fowleri for about 13 hours at about 37° C. using theLDH cytotoxicity assay (infection ratio of 1 amoeba cell per 5-10 CHOcells) as described above. The results are demonstrated in FIG. 10.Protection from high levels of N. fowleri cytotoxicity in vivo wasobserved for Apocynin (IC₅₀=15 μM), Allopurinol (IC₅₀=48 μM), DPI(IC₅₀=49 μM), and hypoxanthine (IC₅₀=497 μM).

Example 7 ROS and Hydrogen Peroxide Production in N. fowleri

Production of reactive oxygen species (ROS) or hydrogen peroxide (H₂O₂)by in vitro cultures of N. fowleri amoebae was measured by Lucigeninassay (50 μM) at 505 nm absorbance for ROS production or by Luminolassay (1 mM) at 425 nm absorbance for H₂O₂ production, respectively.Candida albicans and Leishmania mexicana cultures were used as controls.Results are demonstrated in FIGS. 11A and 11B.

High levels of ROS production characteristic for xanthine oxidase-likeenzymes were detected in live N. fowleri amoebae using the pathogenicfungus Candida albicans and the protozoan parasite Leishmania mexicanafor comparison (Lucigenin assay at 50 μM, Abs 505 nm). In contrast, nosignificant levels of H₂O₂ production were detected in live N. fowleriamoebae or C. albicans (Luminol assay at 1 mM, Abs 425 nm).

We claim:
 1. A method of reducing the pathogenicity of Naegleria fowleriin a subject in need thereof, the method comprising administering atherapeutically effective amount of apocynin to the subject in needthereof.
 2. The method of claim 1, wherein the therapeutically effectiveamount of apocynin reduces cytotoxicity in the subject in need thereof.3. The method of claim 1, wherein the therapeutically effective amountof apocynin reduces the amount of reactive oxygen species in the subjectin need thereof.
 4. The method of claim 1, wherein the therapeuticallyeffective amount of apocynin reduces the oxidoreductase activity of aNfa-1 protein in Naegleria fowleri.